Difference between revisions of "Part:BBa K5492720"

Revision as of 10:09, 2 October 2024

Y1_aptamer_with_fluorophore

Y1 ssDNA aptamer sequence designed to specifically bind to histamine. The sequence contains a fluorophore molecule at its 5' end (5' Cy5™). y1_reverse_compliment_strand is complementary to this sequence, which contains a quencher molecule (3'Iowa Black® RQ) on its 3' end, which absorbs the light emitted by fluorophore until the aptamer binds histamine.

Proposed Folding:

Usage and Biology

Aptamers are generally artificial ssDNA, RNA, or peptide oligomers which bind to specific target molecules. All ssDNA aptamers we utilise are proven to be able to bind specifically to histamine, thus preventing the binding of the molecule to a histamine receptor.

Experiments

Transdermal device

The transdermal device and its usage are written in our protocol in the Experiment topic under the title “Transdermal transport of the enzymes and aptamers”. Please refer that paper for details. Here we emphasize only the final measurement which is a truly simple A260 measurement. We received back from our team members three series of 300 uL volume samples which contained the aptamers packaged in liposomes and dispersed in the hydrogel. We also received two series dispersed in hydrogel without packaging them in liposomes. Each sample series consists of six 300 uL samples arriving to the acceptor phase after 1, 2, 4, 8, 12 and 24 hours.

Before the transdermal experiment we dissolved altogether 552.7 ug DNA aptamers dissolved in 1200 uL TE buffer. From this amount 200 uL was used and was evenly distributed among the five, one-day-long (24 h) transdermal experiment. This means that in each of the five experiments we used 1/5*(200/1200)*552.7 = 18.42 ug aptamers at the donor phase. We determined the DNA content of the acceptor phase by measuring A260 values with Thermofisher Nanodrop device. The results were the following:

The following graph shows the 5 data series results:

As it is clearly visible there are no precise trends in these results. Though the second experiment shows a large peak after eight hours, the other two control experiments don’t represent the same, so we can conclude that there is no evidence for a trend-like transdermal transport amongst aptamers in these experiments. It is still worth it to consider the summed-up transmission:

Considering the exact, total amount of the transmission, we can recognize that only 3-6 ‰ of the aptamers went through the ex vivo membrane, which means that this method is not proper topical use of the aptamers.